The Subcellular Localization Of Ataxin-3and Its Effects On The Morphology Of Cytoplasmic Organoids

OBJECTIVE:In order to better understand the biological function of ataxin-3and the possible toxic mechanism of mutant ataxin-3protein, this study will clarify the subcellular localization of wide type and mutant ataxin-3and the effects of polyQ expansion mutation of ataxin-3on the morphology of mitochondrion, golgi apparatus and endoplasmic reticulum.METHODES:Firstly, pEGFP-N1-MJD20and pEGFP-N1-MJD68, the vectors expressing GFP-ataxin-3fusion proteins,was transfected into Hela cells with Lipofectamine2000. Secondly, twenty-four hours later indirect immunofluorescence was used to identify mitochondria marker TOM20, golgi apparatus marker GM130and endoplasmic reticulum marker calnexin respectively,which were stained with red fluorescent dye. Thirdly, observe whether the ataxin-3proteins were co-localized with cytoplasmic organoids and the effects of polyQ expansion mutation of ataxin-3on the morphology of cytoplasmic organoids with the laser scanning confocal microscope.RESULTS:1. No co-localization was observed between ataxin-3protein and the mitochondrial marker TOM20, but the percentage of cells with mitochondrial fragmentation increased in cells expressing polyQ-expanded ataxin-3(P<0.05).2. No co-localization was observed between ataxin-3protein and the golgi marker GM130and mutant ataxin-3did not cause the golgi fragmentation. 3.Wide type and polyQ-expanded ataxin-3both showed partial co-localization with ER marker calnexin. The latter showed more overlap with calnexin and overlapping signals were mostly located in the places where aggregates were situated.CONCLUSIONS:1. PolyQ-expanded ataxin-3protein may participate in the pathogenesis of SCA3/MJD through indirect damage of mitochondrial integrity.2. PolyQ-expanded ataxin-3may cause no effects on the structure and functions of golgi apparatus.3. Endoplasmic reticulum may be a place where extended ataxin-3protein induces cytotoxicity outside the nucleus.